This invention relates to processes for preparing C-4xe2x80x3 substituted derivatives of 9-deoxo-9a-aza-9a-homoerythromycin A (hereinafter, xe2x80x9cazalide(s)xe2x80x9d) that are useful as antibacterial and antiprotozoal agents in mammals, including man, as well as in fish and birds. This invention also relates to processes for preparing stable intermediates of the subject azalides, as well as to a crystalline salt of an intermediate in the process for preparing the subject azalides. This invention also relates to pharmaceutical compositions containing the novel compounds made by the subject processes and to methods of treating bacterial infections and protozoa infections in mammals, fish and birds by administering the novel compounds produced by the subject processes to mammals, fish and birds requiring such treatment.
Macrolide antibiotics are known to be useful in the treatment of a broad spectrum of bacterial infections and protozoa infections in mammals, fish and birds. Such antibiotics include various derivatives of erythromycin A such as azithromycin which is commercially available and is referred to in U.S. Pat. Nos. 4,474,768 and 4,517,359, both of which are incorporated herein by reference in their entirety. Like azithromycin and other macrolide antibiotics, the macrolide compounds of the present invention possess potent activity against various bacterial infections and protozoa infections as described below.
The production of the subject azalides at commercial scale has presented several difficulties, including, but not limited to, poor yields and instability of some synthetic intermediates, as well as the presence of undesirable impurities.

The present invention relates to a process for preparing a compound of the formula 1
or a pharmaceutically acceptable salt thereof, which comprises: 
reacting a compound of formula 2
with an amine of the formula HNR8R15, in an organic solvent comprising isopropanol;
wherein the reaction is carried out at a temperature of at least about 40xc2x0 C.;
wherein:
R3 is xe2x80x94CH2NR8R15;
R8 is C1-C10 alkyl; and
R15 is H or C1-C10 alkyl.
In a preferred embodiment of the process, R8 is propyl and R15 is H. In a particularly preferred embodiment, R8 is n-propyl and R15 is H.
In a particularly preferred embodiment, the organic solvent is isopropanol.
In another preferred embodiment, the invention relates to a process for 
preparing a compound of the formula 1a or a pharmaceutically acceptable salt thereof,
by reacting a compound of formula 2 with n-propylamine in an organic solvent comprising isopropanol; wherein the reaction is carried out at a temperature of at least about 40xc2x0 C. In a particularly preferred embodiment thereof, the organic solvent is isopropanol.
It is to be noted that the terms xe2x80x9csolutionxe2x80x9d and xe2x80x9cmixturexe2x80x9d, as used herein, unless otherwise indicated, are used interchangeably without regard to the state of dispersion of the components thereof. The phrase xe2x80x9corganic solvent comprising isopropanolxe2x80x9d as used herein, unless otherwise indicated, means a non-aqueous solvent or mixture of non-aqueous solvents, wherein at least one solvent is isopropanol. In this application, the term xe2x80x9ccompound of formula 1xe2x80x9d includes both the compound of formula 1 and the compound of formula 1a. The compound of formula 1a is a particularly preferred embodiment of the compound of formula 1, to which all of the embodiments and preferred embodiments of the processes described herein apply.
In an embodiment of the processes described herein, the temperature is less than about 95xc2x0 C., and in a preferred embodiment thereof, the temperature is less than about 80xc2x0 C. In a more preferred embodiment thereof, the temperature is from about 50xc2x0 C. to about 76xc2x0 C. In a particularly preferred embodiment thereof, the temperature is from about 50xc2x0 C. to about 55xc2x0 C.
In a preferred embodiment of the processes described herein, the reaction is carried out at about atmospheric pressure. In this application, the term xe2x80x9catmospheric pressurexe2x80x9d means a pressure within the normal range of meteorologic atmospheric pressure for a particular altitude, while the term xe2x80x9celevated pressurexe2x80x9d means a pressure above atmospheric pressure. In another embodiment of the processes described herein, the reaction is carried out at elevated pressure. In another embodiment of the invention, triethylamine may be present in addition to isopropanol.
In addition to applicants"" preferred embodiments, the reaction of the compound of formula 2 with an amine to produce the compound of formula 1 has been successfully performed in solvents other than those comprising isopropanol. Accordingly, this invention also relates to a method for preparing a compound of formula 1 by reacting a compound of formula 2 with an amine of the formula HNR8R15, in an organic solvent, wherein the solvent is selected from the group consisting of benzyl alcohol, acetone, methylisobutylketone, DMSO, t-butanol, n-butanol, diisopropylether, a mixture of MTBE and DMF, and combinations thereof, wherein the reaction is carried out at a temperature of at least about 40xc2x0 C. The reaction may be carried out at elevated pressures, but is preferably carried out at about atmospheric pressure. In a further embodiment thereof, the reaction is accelerated by the addition of a catalytic amount of a Lewis acid. In an embodiment thereof, the Lewis acid is a reagent such as magnesium bromide, potassium iodide, lithium perchlorate, magnesium perchlorate, lithium tetrafluoroborate, pyridinium hydrochloride, or tetrabutylammonium iodide. Preferably, the Lewis acid is magnesium bromide.
In an embodiment of the processes described herein, the molar amount of amine is at least about five times the molar amount of the compound of formula 2. In another embodiment of the processes described herein, the concentration of amine in isopropanol is at least about 5 molal. In a particularly preferred embodiment, the concentration of n-propylamine is approximately 6-7 molal in isopropanol.
In an embodiment of the above processes, the compound of formula 2 is reacted with the amine for at least about 24 hours. In a preferred embodiment thereof, the molar amount of the amine is at least about five times the molar amount of the compound of formula 2 and the compound of formula 2 is reacted with the amine for at least about 24 hours. In a more preferred embodiment thereof, the temperature is from about 50xc2x0 C. to about 80xc2x0 C. In a still more preferred embodiment thereof, the molar amount of the amine is about twenty times the molar amount of the compound of formula 2, the concentration of amine in isopropanol is about 6 molal, and the compound of formula 2 is reacted with the amine for at least about 24 hours at a temperature of from about 50xc2x0 C. to about 55xc2x0 C.
Another embodiment of the processes described herein further comprises crystallizing the free base form of the compound of formula 1. In an embodiment, the free base form of the compound of formula 1 is crystallized from an aqueous solvent mixture. In a preferred embodiment thereof, the aqueous solvent mixture comprises water and a non-aqueous solvent selected from the group consisting of methanol, ethanol, isopropanol and acetone. In another embodiment the free base form of the compound of formula 1 is crystallized from an organic (C6-C10)alkane solvent or mixture of such organic alkane solvents. In a preferred embodiment thereof, the compound of formula 1 is crystallized by heating the compound together with the alkane solvent followed by cooling to effect crystallization. In a preferred embodiment thereof, the organic (C6-C10)alkane solvent is selected from heptane or octane, most preferably heptane. In another embodiment, as described below, the free base is prepared from an acid addition salt of the compound of formula 1. It is to be understood that xe2x80x9calkanexe2x80x9d as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbons having straight, cyclic or branched moieties, or mixtures thereof.
In a further embodiment of the processes described herein, an acid addition salt of the compound of formula 1 is prepared by treating the compound of formula 1 with a solution comprising an acid in a water-miscible solvent. In a preferred embodiment thereof, the acid solution is added to a solution comprising the compound of formula 1 and water. In a more preferred embodiment thereof, the acid is phosphoric acid, L-tartaric acid or dibenzoyl-D-tartaric acid. In a particularly preferred embodiment thereof, the acid is phosphoric acid. In another more preferred embodiment thereof, the solvent comprises ethanol. In another preferred embodiment thereof, the above processes further comprise isolating the acid addition salt of the compound of formula 1.
In an embodiment, the processes described herein produce a compound of formula 1 which is at least 90% pure, more preferably at least 95% pure, and most preferably at least 98% pure. In particular, the processes of the invention produce a compound of formula 1 having a purity profile suitable for use of the compound of formula 1 in the preparation of formulations for parenteral administration. The requirements of parenteral formulations are well-known in the art, e.g.: exceptional purity and small particle size in solution, and formulated for sterility and the elimination of pyrogens (see, Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th Edition, Gennaro, ed. (1990), pages 1545-1580.
In another preferred embodiment thereof, the above processes further comprise treating the acid addition salt of the compound of formula 1 with a base in a mixture of water and a nonpolar solvent, to yield the free base form of the compound of formula 1. In a more preferred embodiment thereof, the base is a dibasic carbonate salt, and in a particularly preferred embodiment, the dibasic carbonate salt is potassium carbonate. In another more preferred embodiment thereof, the nonpolar solvent is dichloromethane. In still another embodiment, the process further comprises crystallization of the free base form of the compound of formula 1 as described above, and the further embodiments relating thereto which are described above.
This invention also relates to a process for preparing a compound of formula 2
which comprises:
(a) reacting the free base form of a compound of formula 3
with a sulfonium methylide ion;
(b) quenching the reaction of step (a) with an aqueous weak acid and partitioning the product into a non-aqueous solution; and
(c) deprotecting the product of step (b) to yield the compound of formula 2;
wherein R4 is a hydroxy-protecting group.
In an embodiment, the above process further comprises isolation of the compound of formula 2. In a preferred embodiment thereof, the compound of formula 2 is isolated in the form of a hydrate, more preferably, the monohydrate. In an embodiment thereof, the water content is determined by the Karl-Fischer method. In an embodiment thereof, the hydrate is obtained from a mixture containing the compound of formula 2 and a solvent or solvent mixture selected from acetone, acetone/water, acetone/heptane and MTBE/heptane. In other embodiments, the compound of formula 2 is isolated as its acetate salt, L-tartrate salt or dibenzoyl-D-tartrate salt.
This invention relates to the monohydrate of the compound of formula 2. In a preferred embodiment of the above process, R4 is benzyloxycarbonyl.
In another preferred embodiment of the above process, step (a) is carried out at a temperature of from about xe2x88x9280xc2x0 C. to about xe2x88x9245xc2x0 C.
In a another embodiment, the above process, the free base form of the compound of formula 3 is prepared from an acid addition salt of the compound of formula 3. In a preferred embodiment thereof, the acid addition salt is a trifluoroacetic acid addition salt. In other embodiments of the above processes, the acid addition salt of the compound of formula 3 is selected from a dibenzoyl-D-tartrate salt, a L-tartrate salt, or a phosphate salt. The acid addition salts of the compounds disclosed herein are readily prepared by conventional means.
In an embodiment of the above process, the sulfonium methylide is dimethylsulfonium methylide. In a preferred embodiment thereof the dimethylsulfonium methylide is prepared by reacting a trimethylsulfonium halide or sulfonate with a strong base. In a more preferred embodiment thereof, a trimethylsulfonium halide is used, which is preferably trimethylsulfonium bromide. In another more preferred embodiment thereof, the trimethylsulfonium halide is reacted with the strong base in an inert organic solvent or mixtures thereof. In a particularly preferred embodiment thereof, the inert organic solvent is an ether solvent, which is most preferably tetrahydrofuran, or a mixture of tetrahydrofuran and dichloromethane.
In an embodiment, step (c) comprises catalytic hydrogenation where R4 is benzyloxycarbonyl. In a preferred embodiment thereof, the catalyst for the hydrogenation is a palladium/carbon catalyst. In a particularly preferred embodiment, the palladium/carbon catalyst is 10% Pd/C (Johnson-Matthey type A402028-10). In a further embodiment of step (c), the product of step (b) is deprotected by catalytic transfer hydrogenation, preferably with ammonium formate, Pd/C in methanol. In a further embodiment, the product of step (b) is trated with Fuller""s earth prior to hydrogenation. Suitable solvents for the hydrogenation process are acetone, ethyl acetate, THF, MTBE, isopropanol, ethanol and methanol. A preferred solvent is acetone.
This invention also relates to the 2xe2x80x2-benzyloxycarbonyl protected compound II: 
which is obtained by omitting step (c) of the above processes. 
This invention relates to a process for preparing a compound of formula 3
by oxidation of the C-4xe2x80x3 hydroxy group of a compound of formula 4 
wherein R4 is a hydroxy protecting group.
In an embodiment, the oxidation is performed by adding dimethylsulfoxide (xe2x80x9cDMSOxe2x80x9d) to a solution comprising the compound of formula 4 and a solvent, cooling the mixture to about xe2x88x9270xc2x0 C., and then adding trifluoroacetic anhydride, followed by addition of triethylamine. In other embodiments, the DMSO is activated using oxalyl chloride (with or without trimethylsilylacetamide), polyphosphoric acid, pyridine.SO3, or acetic anhydride. In a further embodiment thereof, the temperature is maintained between xe2x88x9270xc2x0 C. and xe2x88x9260xc2x0 C. during the addition of trifluoroacetic anhydride. In another embodiment thereof, the solvent is dichloromethane. A particular advantage of the above process is the in situ activation of DMSO in the presence of the reacting alcohol, which avoids the formation of impurities typically encountered in activated DMSO oxidations, which usually involve the introduction of the alcohol to a solution containing activated DMSO.
In an embodiment, the above process further comprises isolating an acid addition salt of the compound of formula 3. In a preferred embodiment the acid addition salt is a dibenzoyl-D-tartrate salt or a phosphate salt. In a particularly preferred embodiment, this invention relates to a process for preparing the trifluoroacetic acid addition salt of a compound of formula 3 which comprises treating the compound of formula 3 with trifluoroacetic acid; and crystallizing the resulting acid addition salt;
wherein R4 is a hydroxy-protecting group.
In a preferred embodiment of the above process, R4 is benzyloxycarbonyl.
In another preferred embodiment of the above process, the acid addition salt is crystallized from isopropanol.
In still another preferred embodiment of the above process, the acid addition salt is crystallized from a mixture of methylene chloride and methyl tert-butyl ether.
The trifluoroacetic acid addition salts prepared by the processes of this invention are not pharmaceutically acceptable, but provide excellent purification and stability, allowing the storage and transport of appropriate starting materials in the commercial preparation of compounds of formula 1.
In an embodiment of the above process, the compound of formula 4 is prepared by protection of the 2xe2x80x2-hydroxy group of the compound of formula 5
In a preferred embodiment, the 2xe2x80x2-hydroxy group is protected with benzyloxycarbonyl. In another preferred embodiment, the compound of formula 5 is reacted with at least two molar equivalents of benzylchloroformate. In a more preferred embodiment, the reaction is carried out in dichloromethane. In a still more preferred embodiment, the dichloromethane is present in at least a 15-fold excess volume relative to the volume of starting material. This invention also relates to a 
trifluoroacetic acid addition salt of the compound of formula 3, wherein R4 is benzyloxycarbonyl:
In a preferred embodiment thereof, the salt has the structure shown in formula 3a 
wherein R4 is benzyloxycarbonyl.
This invention also relates to a dibenzoyl-D-tartrate salt of the compound of formula 3, wherein, R4 is benzyloxycarbonyl.
The term xe2x80x9chydroxy-protecting groupxe2x80x9d, as used herein, unless otherwise indicated, includes acetyl, benzyloxycarbonyl, and various hydroxy-protecting groups familiar to those skilled in the art include the groups referred to in T. W. Greene, P. G. M. Wuts, xe2x80x9cProtective Groups In Organic Synthesis,xe2x80x9d (J. Wiley and Sons, 1991). Preferably, the hydroxy-protecting group R4 is benzyloxycarbonyl (xe2x80x9cCBZxe2x80x9d).
The term xe2x80x9chaloxe2x80x9d, as used herein, unless otherwise indicated, includes fluoro, chloro, or bromo, and the term xe2x80x9chalidexe2x80x9d refers to the corresponding mono anions, Fxe2x88x92, Clxe2x88x92, or Brxe2x88x92, respectively.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, cyclic or branched moieties, or mixtures thereof.
The phrase xe2x80x9cpharmaceutically acceptable salt(s)xe2x80x9d, as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. The compounds prepared by the processes of the present invention that are basic in nature, particularly e.g., the free base form of the compounds of formula 1, are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of the present invention are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate)] salts. The compounds prepared by the processes of the present invention that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
The term xe2x80x9ctreatmentxe2x80x9d, as used herein, unless otherwise indicated, includes the treatment or prevention of a bacterial infection or protozoa infection as provided in the method of the present invention.
The present invention includes the compounds of the present invention, and the pharmaceutically acceptable salts thereof, wherein one or more hydrogen, carbon, nitrogen or other atoms are replaced by isotopes thereof. Such compounds may be useful as research and diagnostic tools in metabolism pharmacokinetic studies and in binding assays.